Coupling of the Muscarinic m2 Receptor to G Protein-activated K+ Channels via Gαz and a Receptor-Gαz Fusion Protein
2000; Elsevier BV; Volume: 275; Issue: 6 Linguagem: Inglês
10.1074/jbc.275.6.4166
ISSN1083-351X
AutoresDmitry Vorobiov, Amal Kanti Bera, Tal Keren‐Raifman, Rachel Barzilai, Nathan Dascal,
Tópico(s)Neuropeptides and Animal Physiology
ResumoG protein-activated K+ channel (GIRK), which is activated by the Gβγ subunit of heterotrimeric G proteins, and muscarinic m2 receptor (m2R) were coexpressed in Xenopus oocytes. Acetylcholine evoked a K+ current, I ACh, via the endogenous pertussis toxin (PTX)-sensitive Gi/o proteins. Activation of I ACh was accelerated by increasing the expression of m2R, suggesting a collision coupling mechanism in which one receptor catalytically activates several G proteins. Coexpression of the α subunit of the PTX-insensitive G protein Gz, Gαz, induced a slowly activating PTX-insensitive I ACh, whose activation kinetics were also compatible with the collision coupling mechanism. When GIRK was coexpressed with an m2R·Gαz fusion protein (tandem), in which the C terminus of m2R was tethered to the N terminus of Gαz, part of I ACh was still eliminated by PTX. Thus, the m2R of the tandem activates the tethered Gαz but also the nontethered Gi/o proteins. After PTX treatment, the speed of activation of the m2R·Gαz-mediated response did not depend on the expression level of m2R·Gαz and was faster than when m2R and Gαz were coexpressed as separate proteins. These results demonstrate that fusing the receptor and the Gα strengthens their coupling, support the collision-coupling mechanism between m2R and the G proteins, and suggest a noncatalytic (stoichiometric) coupling between the G protein and GIRK in this model system. G protein-activated K+ channel (GIRK), which is activated by the Gβγ subunit of heterotrimeric G proteins, and muscarinic m2 receptor (m2R) were coexpressed in Xenopus oocytes. Acetylcholine evoked a K+ current, I ACh, via the endogenous pertussis toxin (PTX)-sensitive Gi/o proteins. Activation of I ACh was accelerated by increasing the expression of m2R, suggesting a collision coupling mechanism in which one receptor catalytically activates several G proteins. Coexpression of the α subunit of the PTX-insensitive G protein Gz, Gαz, induced a slowly activating PTX-insensitive I ACh, whose activation kinetics were also compatible with the collision coupling mechanism. When GIRK was coexpressed with an m2R·Gαz fusion protein (tandem), in which the C terminus of m2R was tethered to the N terminus of Gαz, part of I ACh was still eliminated by PTX. Thus, the m2R of the tandem activates the tethered Gαz but also the nontethered Gi/o proteins. After PTX treatment, the speed of activation of the m2R·Gαz-mediated response did not depend on the expression level of m2R·Gαz and was faster than when m2R and Gαz were coexpressed as separate proteins. These results demonstrate that fusing the receptor and the Gα strengthens their coupling, support the collision-coupling mechanism between m2R and the G proteins, and suggest a noncatalytic (stoichiometric) coupling between the G protein and GIRK in this model system. G protein-coupled receptor G protein activated K+channel (Kir3) muscarinic m2 receptor pertussis toxin acetylcholine Members of the Gi/o family of heterotrimeric G proteins (Gi1, Gi2, Gi3, Go1, Go2, and Gz) regulate numerous effectors such as adenylyl cyclase, ion channels, protein kinases, etc. (1.Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar, 2.Gudermann T. Kalkbrenner F. Schultz G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 429-459Crossref PubMed Scopus (333) Google Scholar, 3.Birnbaumer L. Cell. 1992; 71: 1069-1072Abstract Full Text PDF PubMed Scopus (375) Google Scholar, 4.Wickman K. Clapham D.E. Physiol. Rev. 1995; 75: 865-885Crossref PubMed Scopus (344) Google Scholar, 5.Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (603) Google Scholar). A great number of heptahelical G protein-coupled receptors (GPCRs)1 activate Gi/o proteins while usually being rather ineffective in interacting with the other three large families (Gs, Gq, and G12) (2.Gudermann T. Kalkbrenner F. Schultz G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 429-459Crossref PubMed Scopus (333) Google Scholar, 6.Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1576) Google Scholar). The diversity of Gi/o-coupled GPCRs and of the Gαi/o subunits suggests that the various GPCRs should specifically activate different cellular responses via different Gi/o proteins. Many outstanding examples of specific regulation of effectors by certain Gi/o-coupled GPCRs have been described in vivo; however, only limited specificity is observed on the level of receptor-Gαi/o interaction in intact cells and especially in model expression systems (for review see Ref. 2.Gudermann T. Kalkbrenner F. Schultz G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 429-459Crossref PubMed Scopus (333) Google Scholar). It is widely accepted that the overall GPCR-effector coupling specificity is defined by factors such as colocalization or scaffolding of the signaling components, the presence of additional regulatory proteins such as regulators of G proteins signaling, effector/G protein specificity, etc. (6.Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1576) Google Scholar, 7.Neubig R.R. FASEB J. 1994; 8: 939-946Crossref PubMed Scopus (318) Google Scholar, 8.Gudermann T. Schoneberg T. Schultz G. Annu. Rev. Neurosci. 1997; 20: 399-427Crossref PubMed Scopus (251) Google Scholar, 9.Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). However, in the Gi/o-related pathways, these factors still remain poorly understood. One proposed mechanism of ensuring a specific activation of a certain Gα by a GPCR is the existence of a stable complex between the receptor and the G protein in the absence of agonist. The existence of such complexes is supported by several lines of data, among them coimmunoprecipitation of several GPCRs with the corresponding Gα proteins in many cell types (6.Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1576) Google Scholar, 7.Neubig R.R. FASEB J. 1994; 8: 939-946Crossref PubMed Scopus (318) Google Scholar, 8.Gudermann T. Schoneberg T. Schultz G. Annu. Rev. Neurosci. 1997; 20: 399-427Crossref PubMed Scopus (251) Google Scholar, 9.Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). However, in other systems, a collision coupling-type mechanism (10.Levitzki A. Marbach I. Bar-Sinai A. Life Sci. 1993; 52: 2093-2100Crossref PubMed Scopus (16) Google Scholar, 11.Levitzki A. Science. 1988; 241: 800-806Crossref PubMed Scopus (182) Google Scholar) between GPCRs and the G proteins has been demonstrated. In these systems, the receptor is not a priori coupled to Gα, and the coupling takes place only after the binding of an agonist to the receptor. A receptor activated in this way shuttles between and catalytically activates several G proteins in succession.To study activation and effector coupling of individual Gαi/o proteins in separation from the other members of the family, it is necessary to overcome the problem that each of the Gi/o proteins can be activated by almost any Gi/o-interacting GPCR. Attempts to achieve specific coupling in GPCR-Gα pairs were made by creating GPCR·Gα fusion proteins (12.Medici R. Bianchi E. Di Segni G. Tocchini-Valentini G.P. EMBO J. 1997; 16: 7241-7249Crossref PubMed Scopus (41) Google Scholar, 13.Bertin B. Freissmuth M. Jockers R. Strosberg A.D. Marullo S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8827-8831Crossref PubMed Scopus (110) Google Scholar, 14.Wise A. Milligan G. J. Biol. Chem. 1997; 272: 24673-24678Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). However, in Gi/o-containing GPCR·Gα tandems, the tethered receptor still activates "nearby" nontethered Gαi/o proteins (15.Burt A.R. Sautel M. Wilson M.A. Rees S. Wise A. Milligan G. J. Biol. Chem. 1998; 273: 10367-10375Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This problem has been partly overcome (14.Wise A. Milligan G. J. Biol. Chem. 1997; 272: 24673-24678Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) by utilizing the pertussis toxin (PTX) sensitivity, which is a characteristic of all Gαi/o proteins except Gαz (16.Ho M.K. Wong Y.H. Biol. Signals Recept. 1998; 7: 80-89Crossref PubMed Scopus (20) Google Scholar, 17.Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar). PTX catalyzes ADP-ribosylation of a cysteine near the end of the C terminus, preventing the coupling of the agonist-bound receptor to Gα, the dissociation of Gα and Gβγ subunits, and the activation of their effectors (1.Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar). Changing the C-terminal cysteine to glycine or serine renders Gα insensitive to PTX, but it can still be activated by the receptors (18.Kostenis E. Zeng F.Y. Wess J. Life Sci. 1999; 64: 355-362Crossref PubMed Scopus (22) Google Scholar,19.Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (525) Google Scholar). Thus, after PTX treatment, the GPCR of the tandem containing such a mutant Gαi/o interacts only with the tethered Gα. However, another problem arose. In the best studied case, a tandem of the α2A adrenoreceptor with a PTX-resistant mutant Gαi1(C351G), both the receptor-activated GTPase activity and the coupling to the effector (adenylyl cyclase) were substantially impaired as compared with the wild-type Gαi (20.Carr I.C. Burt A.R. Jackson V.N. Wright J. Wise A. Rees S. Milligan G. FEBS Lett. 1998; 428: 17-22Crossref PubMed Scopus (16) Google Scholar, 21.Sautel M. Milligan G. FEBS Lett. 1998; 436: 46-50Crossref PubMed Scopus (16) Google Scholar). This is not surprising, given the importance of the C-terminal end of Gα in receptor recognition and coupling (18.Kostenis E. Zeng F.Y. Wess J. Life Sci. 1999; 64: 355-362Crossref PubMed Scopus (22) Google Scholar, 19.Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (525) Google Scholar) and the devastating effects of some (although not all) mutations of the C-terminal cysteine on receptor-Gα coupling (22.Garcia P.D. Onrust R. Bell S.M. Sakmar T.P. Bourne H.R. EMBO J. 1995; 14: 4460-4469Crossref PubMed Scopus (96) Google Scholar, 23.Osawa S. Weiss E.R. J. Biol. Chem. 1995; 270: 31052-31058Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar).To avoid the use of mutant Gα and to still be able to compare effector activation by free versus receptor-fused Gαi/o protein, we utilized the naturally PTX-resistant Gαz (24.Fong H.K. Yoshimoto K.K. Eversole-Cire P. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3066-3070Crossref PubMed Scopus (183) Google Scholar, 25.Matsuoka M. Itoh H. Kozasa T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5384-5388Crossref PubMed Scopus (153) Google Scholar) and the Xenopus oocyte expression system. Gαz is activated by a variety of Gαi/o-coupled GPCRs and inhibits adenylyl cyclase like other Gαi/o proteins (reviewed in Refs. 16.Ho M.K. Wong Y.H. Biol. Signals Recept. 1998; 7: 80-89Crossref PubMed Scopus (20) Google Scholar and 17.Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar), but Gαz shows a slower GDP-GTP exchange rate and a very low GTPase activity (17.Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar). Recently, endogenous α-adrenergic receptors have been shown to inhibit N-type Ca2+ channels and to activate the G protein-activated, inwardly rectifying K+channels (GIRK) in a PTX-resistant manner in sympathetic neurons after overexpression of Gαz (26.Jeong S.W. Ikeda S.R. Neuron. 1998; 21: 1201-1212Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Both Ca2+ channel inhibition and GIRK activation are mediated by a direct interaction of these channels with Gβγ normally released from Gαi/o proteins (4.Wickman K. Clapham D.E. Physiol. Rev. 1995; 75: 865-885Crossref PubMed Scopus (344) Google Scholar, 27.Dolphin A.C. J. Physiol. 1998; 506: 3-11Crossref PubMed Scopus (233) Google Scholar, 28.Dascal N. Cell. Signal. 1997; 9: 551-573Crossref PubMed Scopus (266) Google Scholar, 29.Ikeda S.R. Dunlap K. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 131-151Crossref PubMed Google Scholar). The use of GIRK channels expressed in Xenopus oocytes as an assay to study the receptor-G protein-effector coupling has been widely utilized (28.Dascal N. Cell. Signal. 1997; 9: 551-573Crossref PubMed Scopus (266) Google Scholar, 30.Yamada M. Inanobe A. Kurachi Y. Pharmacol. Rev. 1998; 50: 723-757PubMed Google Scholar). It allows a controlled expression of different amounts of proteins under study, simply by injecting different amounts of the encoding RNAs, enabling a quantitative study of various aspects of the coupling mechanism. Furthermore, the binding and unbinding of Gβγ to and from the channel are fast. The rise and decay times of the GIRK current upon agonist application and washout are believed to be limited primarily by the rate of G protein activation (normally, the GDP-GTP exchange at the Gα) and by the rate of GTP hydrolysis by Gα, respectively (28.Dascal N. Cell. Signal. 1997; 9: 551-573Crossref PubMed Scopus (266) Google Scholar, 31.Breitwieser G.E. Szabo G. J. Gen. Physiol. 1988; 91: 469-493Crossref PubMed Scopus (150) Google Scholar, 32.Hille B. Neuron. 1992; 9: 187-195Abstract Full Text PDF PubMed Scopus (385) Google Scholar). Therefore, the kinetics of GIRK currents reflect the kinetics of G protein activation and deactivation. Here, using this system, we demonstrate a collision coupling-type (10.Levitzki A. Marbach I. Bar-Sinai A. Life Sci. 1993; 52: 2093-2100Crossref PubMed Scopus (16) Google Scholar) mechanism in activation of the GIRK by m2R via PTX-sensitive Gi/o proteins and via Gz and a substantial improvement of the efficiency of coupling by tethering m2R and the Gα in tandem. Members of the Gi/o family of heterotrimeric G proteins (Gi1, Gi2, Gi3, Go1, Go2, and Gz) regulate numerous effectors such as adenylyl cyclase, ion channels, protein kinases, etc. (1.Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar, 2.Gudermann T. Kalkbrenner F. Schultz G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 429-459Crossref PubMed Scopus (333) Google Scholar, 3.Birnbaumer L. Cell. 1992; 71: 1069-1072Abstract Full Text PDF PubMed Scopus (375) Google Scholar, 4.Wickman K. Clapham D.E. Physiol. Rev. 1995; 75: 865-885Crossref PubMed Scopus (344) Google Scholar, 5.Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (603) Google Scholar). A great number of heptahelical G protein-coupled receptors (GPCRs)1 activate Gi/o proteins while usually being rather ineffective in interacting with the other three large families (Gs, Gq, and G12) (2.Gudermann T. Kalkbrenner F. Schultz G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 429-459Crossref PubMed Scopus (333) Google Scholar, 6.Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1576) Google Scholar). The diversity of Gi/o-coupled GPCRs and of the Gαi/o subunits suggests that the various GPCRs should specifically activate different cellular responses via different Gi/o proteins. Many outstanding examples of specific regulation of effectors by certain Gi/o-coupled GPCRs have been described in vivo; however, only limited specificity is observed on the level of receptor-Gαi/o interaction in intact cells and especially in model expression systems (for review see Ref. 2.Gudermann T. Kalkbrenner F. Schultz G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 429-459Crossref PubMed Scopus (333) Google Scholar). It is widely accepted that the overall GPCR-effector coupling specificity is defined by factors such as colocalization or scaffolding of the signaling components, the presence of additional regulatory proteins such as regulators of G proteins signaling, effector/G protein specificity, etc. (6.Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1576) Google Scholar, 7.Neubig R.R. FASEB J. 1994; 8: 939-946Crossref PubMed Scopus (318) Google Scholar, 8.Gudermann T. Schoneberg T. Schultz G. Annu. Rev. Neurosci. 1997; 20: 399-427Crossref PubMed Scopus (251) Google Scholar, 9.Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). However, in the Gi/o-related pathways, these factors still remain poorly understood. One proposed mechanism of ensuring a specific activation of a certain Gα by a GPCR is the existence of a stable complex between the receptor and the G protein in the absence of agonist. The existence of such complexes is supported by several lines of data, among them coimmunoprecipitation of several GPCRs with the corresponding Gα proteins in many cell types (6.Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1576) Google Scholar, 7.Neubig R.R. FASEB J. 1994; 8: 939-946Crossref PubMed Scopus (318) Google Scholar, 8.Gudermann T. Schoneberg T. Schultz G. Annu. Rev. Neurosci. 1997; 20: 399-427Crossref PubMed Scopus (251) Google Scholar, 9.Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar). However, in other systems, a collision coupling-type mechanism (10.Levitzki A. Marbach I. Bar-Sinai A. Life Sci. 1993; 52: 2093-2100Crossref PubMed Scopus (16) Google Scholar, 11.Levitzki A. Science. 1988; 241: 800-806Crossref PubMed Scopus (182) Google Scholar) between GPCRs and the G proteins has been demonstrated. In these systems, the receptor is not a priori coupled to Gα, and the coupling takes place only after the binding of an agonist to the receptor. A receptor activated in this way shuttles between and catalytically activates several G proteins in succession. To study activation and effector coupling of individual Gαi/o proteins in separation from the other members of the family, it is necessary to overcome the problem that each of the Gi/o proteins can be activated by almost any Gi/o-interacting GPCR. Attempts to achieve specific coupling in GPCR-Gα pairs were made by creating GPCR·Gα fusion proteins (12.Medici R. Bianchi E. Di Segni G. Tocchini-Valentini G.P. EMBO J. 1997; 16: 7241-7249Crossref PubMed Scopus (41) Google Scholar, 13.Bertin B. Freissmuth M. Jockers R. Strosberg A.D. Marullo S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8827-8831Crossref PubMed Scopus (110) Google Scholar, 14.Wise A. Milligan G. J. Biol. Chem. 1997; 272: 24673-24678Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). However, in Gi/o-containing GPCR·Gα tandems, the tethered receptor still activates "nearby" nontethered Gαi/o proteins (15.Burt A.R. Sautel M. Wilson M.A. Rees S. Wise A. Milligan G. J. Biol. Chem. 1998; 273: 10367-10375Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This problem has been partly overcome (14.Wise A. Milligan G. J. Biol. Chem. 1997; 272: 24673-24678Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) by utilizing the pertussis toxin (PTX) sensitivity, which is a characteristic of all Gαi/o proteins except Gαz (16.Ho M.K. Wong Y.H. Biol. Signals Recept. 1998; 7: 80-89Crossref PubMed Scopus (20) Google Scholar, 17.Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar). PTX catalyzes ADP-ribosylation of a cysteine near the end of the C terminus, preventing the coupling of the agonist-bound receptor to Gα, the dissociation of Gα and Gβγ subunits, and the activation of their effectors (1.Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4682) Google Scholar). Changing the C-terminal cysteine to glycine or serine renders Gα insensitive to PTX, but it can still be activated by the receptors (18.Kostenis E. Zeng F.Y. Wess J. Life Sci. 1999; 64: 355-362Crossref PubMed Scopus (22) Google Scholar,19.Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (525) Google Scholar). Thus, after PTX treatment, the GPCR of the tandem containing such a mutant Gαi/o interacts only with the tethered Gα. However, another problem arose. In the best studied case, a tandem of the α2A adrenoreceptor with a PTX-resistant mutant Gαi1(C351G), both the receptor-activated GTPase activity and the coupling to the effector (adenylyl cyclase) were substantially impaired as compared with the wild-type Gαi (20.Carr I.C. Burt A.R. Jackson V.N. Wright J. Wise A. Rees S. Milligan G. FEBS Lett. 1998; 428: 17-22Crossref PubMed Scopus (16) Google Scholar, 21.Sautel M. Milligan G. FEBS Lett. 1998; 436: 46-50Crossref PubMed Scopus (16) Google Scholar). This is not surprising, given the importance of the C-terminal end of Gα in receptor recognition and coupling (18.Kostenis E. Zeng F.Y. Wess J. Life Sci. 1999; 64: 355-362Crossref PubMed Scopus (22) Google Scholar, 19.Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (525) Google Scholar) and the devastating effects of some (although not all) mutations of the C-terminal cysteine on receptor-Gα coupling (22.Garcia P.D. Onrust R. Bell S.M. Sakmar T.P. Bourne H.R. EMBO J. 1995; 14: 4460-4469Crossref PubMed Scopus (96) Google Scholar, 23.Osawa S. Weiss E.R. J. Biol. Chem. 1995; 270: 31052-31058Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To avoid the use of mutant Gα and to still be able to compare effector activation by free versus receptor-fused Gαi/o protein, we utilized the naturally PTX-resistant Gαz (24.Fong H.K. Yoshimoto K.K. Eversole-Cire P. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3066-3070Crossref PubMed Scopus (183) Google Scholar, 25.Matsuoka M. Itoh H. Kozasa T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5384-5388Crossref PubMed Scopus (153) Google Scholar) and the Xenopus oocyte expression system. Gαz is activated by a variety of Gαi/o-coupled GPCRs and inhibits adenylyl cyclase like other Gαi/o proteins (reviewed in Refs. 16.Ho M.K. Wong Y.H. Biol. Signals Recept. 1998; 7: 80-89Crossref PubMed Scopus (20) Google Scholar and 17.Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar), but Gαz shows a slower GDP-GTP exchange rate and a very low GTPase activity (17.Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar). Recently, endogenous α-adrenergic receptors have been shown to inhibit N-type Ca2+ channels and to activate the G protein-activated, inwardly rectifying K+channels (GIRK) in a PTX-resistant manner in sympathetic neurons after overexpression of Gαz (26.Jeong S.W. Ikeda S.R. Neuron. 1998; 21: 1201-1212Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Both Ca2+ channel inhibition and GIRK activation are mediated by a direct interaction of these channels with Gβγ normally released from Gαi/o proteins (4.Wickman K. Clapham D.E. Physiol. Rev. 1995; 75: 865-885Crossref PubMed Scopus (344) Google Scholar, 27.Dolphin A.C. J. Physiol. 1998; 506: 3-11Crossref PubMed Scopus (233) Google Scholar, 28.Dascal N. Cell. Signal. 1997; 9: 551-573Crossref PubMed Scopus (266) Google Scholar, 29.Ikeda S.R. Dunlap K. Adv. Second Messenger Phosphoprotein Res. 1999; 33: 131-151Crossref PubMed Google Scholar). The use of GIRK channels expressed in Xenopus oocytes as an assay to study the receptor-G protein-effector coupling has been widely utilized (28.Dascal N. Cell. Signal. 1997; 9: 551-573Crossref PubMed Scopus (266) Google Scholar, 30.Yamada M. Inanobe A. Kurachi Y. Pharmacol. Rev. 1998; 50: 723-757PubMed Google Scholar). It allows a controlled expression of different amounts of proteins under study, simply by injecting different amounts of the encoding RNAs, enabling a quantitative study of various aspects of the coupling mechanism. Furthermore, the binding and unbinding of Gβγ to and from the channel are fast. The rise and decay times of the GIRK current upon agonist application and washout are believed to be limited primarily by the rate of G protein activation (normally, the GDP-GTP exchange at the Gα) and by the rate of GTP hydrolysis by Gα, respectively (28.Dascal N. Cell. Signal. 1997; 9: 551-573Crossref PubMed Scopus (266) Google Scholar, 31.Breitwieser G.E. Szabo G. J. Gen. Physiol. 1988; 91: 469-493Crossref PubMed Scopus (150) Google Scholar, 32.Hille B. Neuron. 1992; 9: 187-195Abstract Full Text PDF PubMed Scopus (385) Google Scholar). Therefore, the kinetics of GIRK currents reflect the kinetics of G protein activation and deactivation. Here, using this system, we demonstrate a collision coupling-type (10.Levitzki A. Marbach I. Bar-Sinai A. Life Sci. 1993; 52: 2093-2100Crossref PubMed Scopus (16) Google Scholar) mechanism in activation of the GIRK by m2R via PTX-sensitive Gi/o proteins and via Gz and a substantial improvement of the efficiency of coupling by tethering m2R and the Gα in tandem. We are grateful to T. Ivanina, D. Singer-Lahat, I. Lotan, and A. Levitzki for the comments on the manuscript.
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